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217 Pages·2017·6.9 MB·English

Title Investigating metabolomic biomarkers of hypoxic ischaemic
encephalopathy
Author(s) Denihan, Niamh Marie
Publication date 2016
Original citation Denihan, N. M. 2016. Investigating metabolomic biomarkers of hypoxic
ischaemic encephalopathy. PhD Thesis, University College Cork.
Type of publication Doctoral thesis
Rights © 2016, Niamh Marie Denihan.
http://creativecommons.org/licenses/by-nc-nd/3.0/
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Embargo lift date 2017-08-30T11:15:14Z
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Investigating Metabolomic Biomarkers of Hypoxic
Ischaemic Encephalopathy

Niamh Marie Denihan

A thesis submitted to the University College Cork in fulfilment of the
requirements for the degree of Doctor of Philosophy.

January 2016

Supervisors: Dr Deirdre Murray, Dr Jennifer Kirwan and
Prof Geraldine Boylan
Head of department: Prof Jonathan Hourihane

Department of Paediatrics and Child Health
National University of Ireland
University College Cork
Table of Contents
Declaration ............................................................................................................ i
Dedication ............................................................................................................ ii
Acknowledgements ............................................................................................. iii
Abstract ................................................................................................................ v
1 Introduction .................................................................................................. 1
1.1 Background to thesis ........................................................................................ 2
1.1.1 Aims and objectives ................................................................................. 3
1.1.2 Scope ........................................................................................................ 4
1.1.3 Contribution ............................................................................................. 5
2 Background ................................................................................................... 8
2.1 Hypoxic ischaemic encephalopathy ................................................................. 9
2.1.1 Overview .................................................................................................. 9
2.1.2 Current state of the art .......................................................................... 11
2.2 Biochemical pathophysiology of HIE .............................................................. 14
2.2.1 Normal carbohydrate and energy metabolism ..................................... 14
2.2.2 Hypoxia-ischaemia – The energy deficit ................................................ 17
2.2.3 Injury progression .................................................................................. 19
2.3 Current assessment tools ............................................................................... 21
2.3.1 Clinical and biochemical......................................................................... 21
2.3.2 Early neurological outcome measures................................................... 25
2.3.3 Long-term outcome in HIE ..................................................................... 28
2.4 Metabolomics ................................................................................................ 31
2.4.1 Metabolites, metabolomes and metabolomics ..................................... 31
2.4.2 Experimental strategies ......................................................................... 32
2.4.3 Analytical platforms ............................................................................... 36
2.4.4 Untargeted metabolomic workflow ...................................................... 43
2.5 Metabolomics in a neonatal HIE cohort ........................................................ 46
2.5.1 Study population .................................................................................... 46
2.5.2 Sample and data collection .................................................................... 47
2.6 The emerging role of metabolomics in PA and HIE ........................................ 49
2.6.1 Early work in the field ............................................................................ 50
2.6.2 Animal studies ........................................................................................ 50
2.6.3 Metabolomic profiling in neonates........................................................ 56
2.7 Preanalytical variation in metabolomics ....................................................... 59
2.7.1 Haemolysis ............................................................................................. 60
3 Untargeted metabolomic analysis and pathway discovery in perinatal asphyxia and
hypoxic ischaemic encephalopathy ..................................................................... 63
3.1 Abstract .......................................................................................................... 64
3.2 Introduction .................................................................................................... 65
3.3 Methodology .................................................................................................. 66
3.3.1 Study population .................................................................................... 66
3.3.2 Metabolomic analysis of UCB serum with DI FT-ICR MS ....................... 67
3.3.3 Statistical analysis .................................................................................. 69
3.3.4 Putative metabolite annotation and pathway analysis ......................... 70
3.4 Results ............................................................................................................ 72
3.4.1 Study population .................................................................................... 72
3.4.2 Univariate analysis ................................................................................. 74
3.4.3 Multivariate analysis .............................................................................. 84
3.4.4 Pathway analysis .................................................................................... 87
3.5 Discussion ....................................................................................................... 91
4 Lipid metabolite alterations in perinatal asphyxia and neonatal hypoxic ischaemic
encephalopathy ................................................................................................ 100
4.1 Abstract ........................................................................................................ 101
4.2 Introduction .................................................................................................. 102
4.3 Methodology ................................................................................................ 104
4.3.1 Metabolomic analysis of UCB serum with DI FT-ICR MS ..................... 104
4.3.2 Statistical analysis ................................................................................ 105
4.3.3 Putative metabolite annotation ........................................................... 107
4.4 Results .......................................................................................................... 108
4.4.1 Univariate analysis ............................................................................... 108
4.4.2 Multivariate analysis ............................................................................ 116
4.5 Discussion ..................................................................................................... 118
5 Cord metabolite model as a predictor of outcome in perinatal asphyxia and hypoxic
ischaemic encephalopathy ................................................................................ 124
5.1 Abstract ........................................................................................................ 124
5.2 Introduction.................................................................................................. 126
5.3 Methodology ................................................................................................ 127
5.3.1 Patient selection .................................................................................. 127
5.3.2 Three- year outcome ........................................................................... 128
5.3.3 Metabolomic analysis .......................................................................... 128
5.3.4 Statistical analysis ................................................................................ 129
5.4 Results .......................................................................................................... 131
5.4.1 Study population .................................................................................. 131
5.4.2 Prediction of outcome ......................................................................... 134
5.5 Discussion ..................................................................................................... 143
6 The effect of haemolysis on the metabolomic profile of cord blood .......... 146
6.1 Abstract ........................................................................................................ 147
6.2 Introduction.................................................................................................. 148
6.3 Methodology ................................................................................................ 149
6.3.1 Patient selection and sample collection .............................................. 149
6.3.2 Haemolysis ........................................................................................... 149
6.3.3 Metabolomic analysis .......................................................................... 150
6.3.4 Statistical Analysis ................................................................................ 150
6.4 Results .......................................................................................................... 151
6.5 Discussion ..................................................................................................... 155
7 Characterising the effect of haemolysis on the cord blood metabolome by direct infusion
mass spectrometry ........................................................................................... 158
7.1 Abstract ........................................................................................................ 159
7.2 Introduction.................................................................................................. 160
7.3 Methodology ................................................................................................ 162
7.3.1 Sample selection .................................................................................. 162
7.3.2 Haemolysis ........................................................................................... 162
7.3.3 Metabolomic analysis of UCB serum with DI FTICR MS ...................... 163
7.4 Putative metabolite annotation ................................................................... 166
7.4.1 Data analysis ........................................................................................ 167
7.5 Results .......................................................................................................... 168
7.5.1 Multivariate analysis ............................................................................ 169
7.5.2 Univariate analysis ............................................................................... 172
7.5.3 Comparison to potential biomarkers ................................................... 178
7.6 Discussion ..................................................................................................... 179
8 Concluding Summary ................................................................................ 185
8.1 Summary of main findings ........................................................................... 186
8.1.1 Strengths .............................................................................................. 188
8.1.2 Limitations ............................................................................................ 189
8.2 Impact of thesis ............................................................................................ 191
8.3 Future work .................................................................................................. 192
Bibliography ..................................................................................................... 196
Appendix A. List of Abbreviations .................................................................. 216
Appendix B. List of Figures ............................................................................. 219
Appendix C. List of Tables .............................................................................. 223
Appendix D. Dissemination of Work ............................................................... 226
D.1. Peer-reviewed publications ...................................................................... 226
D.2. Conference presentations ........................................................................ 226
D.3. Other achievements ................................................................................. 228
Appendix E. Cohort identification .................................................................. 230
E.1. Historical prospective cohort - The BIHIVE Study ..................................... 230
E.2. Healthy prospective cohort - The BABYCORD Study ................................ 232
E.2.1 Information and consent form ............................................................. 234
E.2.2 Staff information leaflet ....................................................................... 237
E.2.3 Amendments to ethical approval ........................................................ 238
E.2.4 Data collection sheet ........................................................................... 239
E.3. Prospective validation cohort - The BIHIVE2 Study .................................. 240
E.3.1 Case consent form ............................................................................... 245
E.3.2 Case patient information leaflet .......................................................... 249
E.3.3 Control consent form ........................................................................... 252
E.3.4 Control patient information leaflet ..................................................... 254
Appendix F. Sample biobanking ..................................................................... 257
F.1. Umbilical cord blood (UCB) ...................................................................... 257
F.2. Postnatal samples .................................................................................... 262
F.3. Sample storage and transport ................................................................. 263
Appendix G. Metabolomic analysis................................................................. 264
G.1. Metabolite extraction .............................................................................. 264
G.2. Sample preparation for mass spectrometry ............................................ 267
G.3. FT-ICR mass spectrometry – acquisition of spectra ................................. 268
G.4. Processing mass spectral data ................................................................. 271
G.5. Putative metabolite annotation and pathway analysis ........................... 272
Appendix H. Putatively annotated metabolic features.................................... 274
Appendix I. Metabolic features assigned empirical formulae ............................ 293
Appendix J. Publications arising from thesis ...................................................... 311
Declaration
This is to certify that the work I am submitting is my own and has not been submitted for
another degree, either at University College Cork or elsewhere. All external references and
sources are clearly acknowledged and identified within the contents. I have read and
understood the regulations of University College Cork concerning plagiarism.

Signed: ________________________ Date: _______________
i
Dedication

To
Joe and Rita Hyland

Eithne, Bill and Brian Denihan
ii
Acknowledgements
When I arrived at University College Cork in 2011 to embark on a PhD, I was extremely
privileged to meet Dr Deirdre Murray and Prof Geraldine Boylan whose passion for neonatal
research was infectious and their work clearly outstanding. With their support I was primed to
devote the next four years to a challenging and meaningful PhD project. When, the following
year, I met Dr Jennifer Kirwan, who shared her love and passion for metabolomics, I knew our
project was in safe hands.
So first and foremost I must acknowledge and thank each of my supervisors, Dr Deirdre
Murray, Dr Jennifer Kirwan and Prof Geraldine Boylan. I greatly appreciate you generously
sharing your expertise and providing guidance, as well as being a constant source of
reassurance and optimism. Deirdre and Geraldine, thank you for presenting me with endless
opportunities and encouraging me to seize each one, including opportunities to collaborate,
present, compete and travel. Jennifer, thank you for always being so accessible to me; your
feedback and advice along this journey has been invaluable.

I am lucky and grateful to have been a part of the BIHIVE team, alongside Mrs Jean Conway, Ms
Ann Marie Looney, Dr Brian Walsh and Dr Caroline Ahearne. Our close comradery provided the
motivation to endure and dare I say enjoy the on-call sleepless nights and all of the peaks and
pitfalls that go with research. I can’t thank you enough for your help. This thesis may be a very
small step, but I believe our concerted effort will one day make a real impact. I must
acknowledge the midwives and clinical staff of Cork University Maternity Hospital. Without
their dedicated help as well as the generous participation of the parents and children, the
BIHIVE study would not be possible.
A word of appreciation to all of the friends and colleagues I have met along this journey, of
which there are too many to name individually. To those in the Department of Paediatrics and
Child Health, the Irish Centre for Fetal and Neonatal Translational Research (INFANT) and the
Department of Medicine, it has been an education and a pleasure.
I will be forever grateful to Molecular Medicine Ireland (MMI) for awarding me a place on the
Clinical and Translational Research Scholars Programme. Not only was this instrumental in my
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